1. Field of the Invention
The lack of standard levels of pharmacologically active compounds in natural botanical products has resulted in a reluctance by health care providers to prescribe these products for their patients. This problem is compounded by the fact that even when botanical products are standardized to specific levels of a marker compound, variations in the content of other oftentimes unknown compounds caused by a wide range of factors such as soil temperature and pH, air temperature, rainfall, and genetics, results in unpredictable biological activity. Additionally, raw material integrity is vital to the production of a safe and effective product. Therefore, this invention is directed to a method of assuring reproducibility of an extraction process. The present invention is also directed to a method of reproducibly extracting a pharmacologically active mixture of chemical components from a biological source, particularly a plant source. Furthermore, the present invention is directed to a method of manipulating the pharmacologically active compound concentration levels within said pharmacologically active mixture of chemical compounds relative to certain other unknown compounds within the mixture. Thus, the method delivers a pharmaceutical grade product with a desired level of bioactivity without adulteration of any kind. The present invention also reveals a composition of botanical material with enhanced biological activity when compared with the individual parts or the sum of the parts.
2. Background of the Invention
Plants have been, and continue to be, the primary source of a wide variety of medicinal compounds. For centuries, various forms of botanically derived materials have been used to treat countless different ailments. The botanical materials have typically been in the form of powders made from one or more plants or plant parts or extracts derived from whole plants or selected plant parts. These powders and extracts are, for the most part, complex mixtures of both biologically active and biologically inactive compounds.
In modern day, herbal medicine, although gaining some acceptance in Western society, still faces several specific challenges. First, in the opinion of many highly trained medical practitioners, there is the view that herbal medicine lacks sufficient scientific support data in our highly technical and science oriented society. Secondly, there is concern about which components of an herbal remedy are pharmaceutically effective. Furthermore, the question arises as to the concentrations or dosages present of such pharmaceutically effective components of herbal remedies. In short, traditional medical practitioners are concerned with a lack of both qualitative and quantitative standards for herbal medications. Such a lack of standardization is viewed as hindering the ability to prescribe and adjust dosages of such nontraditional or herbal medications. The lack of such standardization has also lead to a reluctance on the part of regulatory agencies in funding further investigation and acceptance of such nontraditional medications.
Although not meeting some of the criteria of Western traditional medicine, such herbal compositions are known to be quite effective in treatment of a variety of maladies with little or no side effects. In part, the pharmaceutical activity in many instances is attributable not only to the presence of specific biologically active compounds but also to a synergistic effect resulting from the combination of two or more chemical components present in the herbal mixture.
Since herbal treatments, defined as both herbal medications and biologically enhancing herbal compositions, are derived from plants, the chemical composition of such herbal treatments varies according to a number of factors, not the least of which are the genetic composition and growing conditions in which the plant is produced as well as the harvest conditions and isolation of the active components of the plant.
Accordingly, biological variants of a particular plant may typically be expected to produce significant variations in quantities of particular chemical components found in the plant. Likewise, even in the same biological variant of a plant, differences in soil, moisture and other growing conditions may significantly affect the quantities of specific chemical components produced by the plant.
The vast majority of the raw material used to produce botanical products is currently collected form the wild. Wild collected plants are, by definition, produced outside of a controlled environment. This has presented a unique problem for manufacturers of botanical products desiring the control, reproducibility, and standardization that are required of pharmaceuticals. This problem is due primarily to the plurality of components contained in an herbal medicine and the large variation in composition and potency due to the growing, harvesting and processing conditions. Therefore it is desirable to standardize these growing conditions to the extent possible.
Finally, the manner in which a plant is processed can drastically influence the relative proportions and total amounts of specific chemical components isolated from the plant. Thus, such steps as harvesting, storage, reduction in particle size, expression of liquid components and extraction all determine the proportions and amounts of chemical components and hence the pharmaceutical activity of the isolated product.
Considering the many factors which influence the composition and pharmaceutical activity of herbal compositions, it is desirable to employ methods which result in the standardization of herbal compositions both with respect to the chemical compositions thereof and the pharmaceutical activity of such chemical mixtures. In addition, it is desirable to standardize the processing conditions in order to obtain such standardized herbal compositions. Furthermore, being able to accurately determine and compare the compositions of biological mixtures, particularly plant or herbal mixtures, would allow processing conditions to be controlled to obtain reliable pharmacological activity. With such methods available to the scientific community, not only would physicians be able to prescribe specified dosages of herbal compositions with confidence, but herbal composition “manufacturers” would achieve predictable pharmaceutical activity of such mixtures, improved quality control and the ability to differentiate herbal mixtures from varying sources.
Pharmaceutical grade botanical products are advantageous in that they allow careful tracking of the effects of individual compounds, in treatment protocols. Further, the dosage of the drug can be carefully controlled to provide relatively predictable medicinal action. The potential benefit provided by the use of herbal product as medicine is believed by many industry experts to be outweighed by the clinical risks associated with the absence of standard levels of biologically active materials from natural plants. In that respect, herbal products are not well characterized or controlled in a clinical setting.
The present invention is a significant advancement in the field of making herbal extracts for medicinal purposes, providing precise levels of both berberine and hydrastine while at the same time maintaining USDA National Organic Program (NOP) organic certification. The invention offers tremendous benefits since the extract is standardized to biological activity rather than just a chemical marker. This allows for the use of exact quantities of the extract necessary to produce the desired pharmacological activity instead of potentially toxic high doses or ineffective low doses produced by current methods. The invention also permits the manipulation and/or removal of certain pharmacologically active compounds from the extract thereby allowing more precise calculation of the contribution of each compound's activity to that of the whole and permits various ratios of the compounds in the final product based on the results of certain bioassays. In addition, pre-standardization by cultivation and harvesting practices results in higher concentrations of pharmacologically active compounds in the raw material which translates into reduced processing to produce a given amount of extract. Maintenance of USDA NOP certification throughout the production process provides third party certification that the product has been produced in a sustainable manner, important due to many medicinal plant's endangered status, and that no potentially toxic materials such as herbicides, pesticides, or artificial fertilizers have been used.
In one object of this invention, a method was developed for standardizing the levels of biologically active materials from natural plants including, but not limited to, herbal plants. In a more preferable object of this invention, the method is suitable for producing pharmaceutical grade natural products standardized to a specific level of bio-activity from Hydrastis canadensis Linn. (goldenseal) and other berberine and/or hydrastine containing plants. In a preferable object of this invention, the method disclosed is suitable for the precise standardization of marker compounds generally recognized as indicators for the quality of goldenseal. In a more preferable object of this invention, the marker compounds are berberine and hydrastine.
Background Information Regarding Goldenseal
Goldenseal belongs to the Family Ranunculaceae and is a small hairy perennial which in a natural environment generally emerges in early spring (mid-March to early May) and dies down in mid-August to late-September. The root system is composed of a bright yellow horizontal rhizome, 2 to ¾ inch thick, marked by cup-like depressions where the annual stem falls away. Mature plants (at least 3 years old) are 6-14 inches tall and have two or more hairy stems usually ending in a fork with two leaves. The 5-7 lobed, palmate, double-toothed leaves are 3-12 inches wide and 3-8 inches long. After emergence in early spring, flower buds quickly develop and small inconspicuous white flowers open as the leaves unfold. Plants started from seed usually flower the third or fourth year. Each plant can produce a single, green raspberry like fruit which turns red and ripens in July (Davis, 2000).
Goldenseal is one of the most popular medicinal herbs in the US and has been wild collected from the forests of Eastern North America for hundreds of years. Its historical range extended from Vermont to Georgia to Arkansas to Minnesota (Foster, 1990). Goldenseal's popularity has resulted in it being overcollected from the wild. Goldenseal is listed as an Endangered Species in Georgia, North Carolina, Vermont, Connecticut, Massachusetts, and Minnesota. The USFWS has stated that goldenseal is either endangered, threatened, imperiled, rare, or uncommon in all 27 U.S. states which have native populations (USFWS, 1997). The US government, in 1997, backed a proposal to place Goldenseal on the Convention on International Trade in Endangered Species (CITES) Appendix List II, which was approved. The CITES listing requires the goldenseal produced for export be cultivated for minimum of four years. The present invention addresses this problem through the development of a “wild simulated” production system for goldenseal.
The alkaloids berberine and hydrastine are commonly used by industry as markers to indicate the quality of raw goldenseal although the most common level of consumer product assurance is that of a specified percentage of total alkaloid content, unspecified as which alkaloid or how much of each. These two compounds are also thought to be the primary bio-active compounds in the plant. (Abourashed, 2001, Govindan, 1999) Proposed United States Pharmacopoeia (USP) standards for dried goldenseal root and rhizome are not less than 2.0% hydrastine and not less than 2.5% berberine.
The alkaloid content variability of both raw goldenseal and consumer products currently on the market was clearly demonstrated in Govindan (1999), where ten samples (eight—raw powdered material, GS-1 through GS-8 and two—powdered material from off-the-shelf capsules, GS-9 and GS-10) were analyzed using Thin-Layer Chromatography (TLC). Five of the samples contained both berberine and hydrastine, four contained only berberine, and one contained neither. These results were verified by High Pressure Liquid Chromatography (HPLC) (Govindan, 1999). Hydrastine is unique to goldenseal among North American plants therefore the absence of hydrastine is a strong indication that the material purported to be goldenseal root and rhizome is probably not goldenseal at all. Extremely low quantities of hydrastine are also indicative of adulteration with other berberine containing plants such as various Berberis species. Discovery of palmatine in purported goldenseal material, common in other berberine producing plants but absent in goldenseal, is a definitive indication of adulteration (Weber, 2003). In addition only three of the ten samples tested approximated the profile of the raw goldenseal used by the National Toxicological Program for their studies (MRI, 2001). Our 2003 harvest (SHF-1) had the second highest combined hydrastine/berberine total of all the samples listed. See table 1 below.
The present invention provides both a method of standardized processing procedures and of obtaining biological compositions having a desired level of pharmaceutical activity from plants containing berberine and/or hydrastine, preferrably goldenseal. The present invention also permits the isolation of biological compositions or components, and in particular herbal compositions or components, having high, or the highest pharmacological activity obtainable by a specific process, such as extraction.
TABLE 1Results of TLC and HPLC analysis of 10 goldenseal samples(GS-1-GS-10)a, National Toxicological Program Sample(NTP-1)b,one commercial product analyzed in Weber, et al, (1999)cand Sleepy Hollow Farms 2003 harvest.HydrastineBerberineHydrastinineSampleTLCHPLC(%)TLCHPLC(%)TLCHPLC(%)GS-1+++n.a.+++n.a+n.a.GS-2+++2.68+++4.27+n.a.GS-3−−++0.56+n.a.GS-4+++2.34+++3.48+n.a.GS-5+++3.22+++4.54+n.a.GS-6+0.60++0.56−n.a.GS-7−−++0.28−n.a.GS-8−−−−−n.a.GS-9−−+++1.51−n.aGS-10−−+++5.31−n.a.NTP-1n.a.3.02n.a.3.45n.a.n.a.Weber-1n.a.1.30n.a.1.90n.a.n.a.SHF-1n.a.3.31n.a.3.96n.a.n.a.aRelative amounts based on the visual estimation of the size and intensity of spots on TLC: dark spot(+++); medium intensity spot(++); low intensity spot(+); not visible(−); not analyzed(n.a.)(Govindan, 1999).b(MRI, 2001)c(Weber, et al., 1999)Use of Goldenseal and/or its Isolated Alkaloids Berberine/Hydrastine
Goldenseal was first used by Native Americans to treat wounds, ulcers, digestive disorders, and skin and eye ailments. Over the years goldenseal has been used to treat a variety of digestive and hemorrhagic disorders. It is thought to possess antiseptic, astringent, and hemostatic qualities when applied topically. It is thought by some that goldenseal is effective in the treatment of diarrhea, hemorrhoids, disorders of the genito-urinary tract, upper respiratory inflammation and congestion, mucous membrane inflammation, eczema, pruritus, otorrhea, tinnitus and congestion/inflammation of the ear, and conjunctivitis, as well as for cancers, particularly of the ovary, uterus, and stomach. Goldenseal has been used as a tonic, antiperiodic, diuretic, and as a vaginal douche. It is commonly consumed in capsules, liquid herbal extracts, and as an herbal tea.
Although at least 10 isoquinoline alkaloids have been identified in goldenseal, berberine is considered to be the primary pharmacologically active compound in goldenseal. Berberine's most common historic and clinical uses include bacterial diarrhea, intestinal parasites, and ocular trachoma infections. Berberine has been shown to exhibit significant antimicrobial activity against a variety of bacteria, fungi, protozoans, helminths, chlamydia, and viruses (Birdsall, 1997). Some berberine containing plants have been shown to produce substances which, by themselves, are completely without antimicrobial activity however, when used in conjunction with berberine, they enhanced the activity of berberine by as much as 2,500 times (Stermitz, 2000, Tegos, 2002). In addition to this antimicrobial activity, berberine has been found to have numerous pharmacological effects including antagonism of the effect of cholera and E. coli heat stable enterotoxin (Sack, 1982), delay of small intestine transit time (Eaker, 1989), inhibition of intestinal secretions (Zhu, 1983), significantly inhibit spontaneous peristalsis in the intestine (Birdsall, 1997), inhibition of smooth muscle contraction (Tai, 1981), potent inflammation inhibitory activity (Yesilada, 2002), inhibition of cyclooxygenase-2 (COX-2) transcriptional activity in a dose and time dependent manner (Fukuda, 1999), and inhibition of II-8 production in rectal mucosa in rats (Zhou, 2000). Furthermore, berberine has been shown to produce significant cardiovascular, cholesterol reduction, MAO inhibition, and antidiabetic activities. Berberine is considered to be a non-antibiotic anti-diarrheal drug (Baird, 1997).
Several recent studies have been conducted regarding the in vitro effectiveness of the antimicrobial constituents of goldenseal crude extract and its isolated alkaloids against a variety of oral and digestive tract pathogens including Streptococcus mutans, Fusobacterium nucleatum, and 15 strains of Helicobacter pylori. Results indicated the crude extract and isolated berberine to be very active against these pathogens (Hwang, 2003, Mahady, 2003).
In a 2001 study, the antibacterial activity of crude goldenseal extract and the isolated alkaloids berberine, hydrastine, and canadine were evaluated against five strains of microorganism: Staphylococcus aureus (ATCC 25 993 and ATCC 6538P), Streptococcus sanguis (ATCC 10 556), Escherichia coli (ATCC 25 922), and Pseudomonas aeruginosa (ATCC 27 853). The results of this study were reported to provide a rational basis for the traditional antibacterial use of goldenseal (Scazzocchio, 2001).
Berberine, isolated from the roots of goldenseal, was demonstrated to be responsible for the significant activity of goldenseal extract against multiple drug resistant Mycobacterium tuberculosis. (Gentry, 1998)
A 1998 study conducted at Iowa State University entitled Botanicals for Pigs—Goldenseal compared goldenseal's use as a natural antimicrobial agent in nursery pigs with Mecadox. The study reported that growth of the pigs on a 1% goldenseal diet were often not statistically different from the Mecadox controls.
One significant thread common to the above studies is the fact that in each instance the activity of the crude extract was shown to be equal to or greater than the activity of isolated berberine. In one particular instance, Hwang (2003), the crude extract of goldenseal root and rhizome was demonstrated to have comparable activity to the isolated berberine even though the crude extract only contained 0.02% berberine, 1/5000th the concentration of the isolated berberine. All other fractions and combinations of alkaloids produced only minimal activity and can not possibly account for the increased activity of the crude extract. This indicates there are other unknown compounds in goldenseal root and rhizome which are inactive by themselves but significantly enhance berberine's activity when combined. Accordingly standardization to specific quantities of berberine in a goldenseal product will most likely produce varying pharmacological activity due to year to year variations in environmental conditions which produce not only fluctuating amounts of berberine but also the unknown compounds.
This invention reveals a method of manipulating the concentration levels of goldenseal's primary marker compounds within the final product without adulteration of any kind. In addition, the invention goes a step further by revealing a method of quantifying the effects of the unknown compounds in goldenseal then using the aforementioned method to produce a goldenseal product which produces a predictable level of pharmacological activity.